U.S. patent number 6,949,584 [Application Number 10/783,986] was granted by the patent office on 2005-09-27 for tnp-470 species, polymer conjugates and use thereof.
This patent grant is currently assigned to Children's Medical Center Corporation. Invention is credited to Judah Folkman, Ronit Satchi-Fainaro.
United States Patent |
6,949,584 |
Satchi-Fainaro , et
al. |
September 27, 2005 |
**Please see images for:
( Certificate of Correction ) ** |
TNP-470 species, polymer conjugates and use thereof
Abstract
The present invention relates to conjugates of water-soluble
polymers and o-(chloracetyl-carbamoyl) fumagillol (TNP-470) and use
of those conjugates as specific intracellular carriers of the
TNP-470 into tumor vessels. The present invention further relates
to use of those conjugates to lower the neurotoxicity of TNP-470.
Preferably, the polymer has a molecular weight in the range of 100
Da to 800 kDa. More preferably, the polymer has a molecular weight
no greater than 60 kDa. Most preferably, the polymer has a
molecular weight in the range of 15 kDa to 40 kDa.
Inventors: |
Satchi-Fainaro; Ronit (Chestnut
Hill, MA), Folkman; Judah (Brookline, MA) |
Assignee: |
Children's Medical Center
Corporation (Boston, MA)
|
Family
ID: |
29254460 |
Appl.
No.: |
10/783,986 |
Filed: |
February 19, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
PCTUS0310976 |
Apr 10, 2003 |
|
|
|
|
Current U.S.
Class: |
514/475;
549/332 |
Current CPC
Class: |
C08L
53/00 (20130101); C07D 303/22 (20130101); A61P
35/04 (20180101); A61K 47/58 (20170801); C08F
220/56 (20130101); A61P 9/00 (20180101); A61P
35/00 (20180101); C08F 220/58 (20130101); C08L
53/00 (20130101); C08L 2666/02 (20130101) |
Current International
Class: |
A61K
31/336 (20060101); A61K 31/335 (20060101); C07D
407/08 (20060101); C07D 305/14 (20060101); C07D
407/00 (20060101); C07D 305/00 (20060101); A61P
35/00 (20060101); A61P 35/04 (20060101); C07D
407/08 (); A61K 031/336 (); A61P 035/04 () |
Field of
Search: |
;514/475 ;549/332 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Folkman, J., Angiogenesis, in Harrison's Textbook of Internal
Medicine (eds. Braunwald, E. et al.) 517-530 (McGraw Hill, New
York, 2001). .
Hanahan, D. et al., Patterns and emerging mechanisms of the
angiogenic switch during tumorigenesis, Cell, 86:353-64 (1996).
.
Volpert, O.V. et al., Id1 regulates angiogenesis through
transcriptional repression of thrombospondin-1, Cancer Cell,
2:473-483 (2002). .
Folkman, J., Tumor angiogenesis, Cancer Medicine (eds. Holland, J.
et al.), pp. 132-152 (B. C. Decker Inc., Ontario, Canada, 2000).
.
Lyden, D. et al., Id1 and Id3 are required for neurogenesis
angiogenesis and vascularization of tumour xenografts, Nature,
401:670-677 (1999). .
Streit, M. et al., Thrombospondin-2: a potent endogenous inhibitor
of tumor growth and angiogenesis, Proc. Natl. Acad. Sci. USA,
96:14888-14893 (1999). .
Chin, L. et al., Essential role for oncogenic Ras in tumour
maintenance, Nature, 400:468-472 (1999). .
Tabone, M.D. et al., Are basic fibroblast growth factor and
vascular endothelial growth factor prognostic indicators in
pediatric patients with malignant solid tumors?, Clinical Cancer
Res., 7:538-543 (2001). .
Yao, Y. et al., Prognostic value of vascular endothelial growth
factor and its receptors Flt-1 and Flk-1 in astrocytic tumours,
Acta Neurochir (Wien), 143:159-66 (2001). .
Yuan, A. et al., Aberrant p53 expression correlates with expression
of vascular endothelial growth factor mRNA and interleukin-8 mRNA
and neoangiogenesis in non-small-cell lung cancer, J. Clinical
Oncology, 20:900-910 (2002). .
Ingber, D. et al., Synthetic analogues of fumagillin that inhibit
angiogenesis and suppress tumour growth, Nature, 348:555-557
(1990). .
Antoine, N. et al., AGM-1470, a potent angiogenesis inhibitor,
prevents the entry of normal but not transformed endothelial cells
into the G.sub.1 phase of the cell cycle, Cancer Res., 54:2073-2076
(1994). .
Kudelka, A.P. et al., Complete remission of metastatic cervical
cancer with the angiogenesis inhibitor TNP-470, N. Engl. J. Med.,
338:991-2 (1998). .
Kudelka, A.P. et al., A phase I study of TNP-470 administered to
patients with advanced squamous cell cancer of the cervix, Clinical
Cancer Res., 3:1501-1505 (1997). .
Bhargava, P. et al., A Phase I and pharmacokinetic study of TNP-470
administered weekly to patients with advanced cancer, Clinical
Cancer Res., 5:1989-1995 (1999). .
Herbst, R.S. et al., Safety and pharmacokinetic effects of TNP-470,
an angiogenesis inhibitor, combined with paclitaxel in patients
with solid tumors: evidence for activity in non-small-cell lung
cancer, J. Clinical Oncol., 20:4440-4447 (2002). .
Kim, E.S. et al., Angiogenesis inhibitors in lung cancer. Curr.
Oncol. Rep., 4:325-333 (2002). .
Stadler, W.M. et al., Multi-institutional study of the angiogenesis
inhibitor TNP-470 in metastatic renal carcinoma, J. Clinical
Oncol., 17:2541-2545 (1999). .
Logothetis, C.J. et al., Phase I trial of the angiogenesis
inhibitor TNP-470 for progressive androgen-independent prostate
cancer, Clinical Cancer Res., 7:1198-1203 (2001). .
Rupnick, M.A. et al., Adipose tissue mass can be regulated through
the vasculature, Proc. Natl. Acad. Sci. U S A, 99:10730-10735
(2002). .
Schoof, D.D. et al., The influence of angiogenesis inhibitor
AGM-1470 on immune system status and tumor growth in vitro, Int. J.
Cancer, 55:630-635 (1993). .
Nagabuchi, E. et al., TNP-470 antiangiogenic therapy for advanced
murine neuroblastoma, J. Pediatric Surg., 32:287-93 (1997). .
Rihova, B. et al., Biocompatibility of N-(2-hydroxypropyl)
methacrylamide copolymers containing adriamycin. Immunogenicity,
and effect on haematopoietic stem cells in bone marrow in vivo and
mouse splenocytes and human peripheral blood lymphocytes in vitro,
Biomaterials, 10:335-342. (1989). .
Seymour, L.W. et al., The pharmacokinetics of polymer-bound
adriamycin, Biochem. Pharmacol., 39:1125-1131 (1990). .
Maeda, H. et al., Tumor vascular permeability and the EPR effect in
macromolecular therapeutics: a review, J. Controlled Release,
65:271-284 (2000). .
Duncan, R. et al., Preclinical toxicology of a novel polymeric
antitumour agent: HPMA copolymer-doxorubicin (PK1), Human and Exp.
Toxicology, 17:93-104 (1998). .
Satchi-Fainaro, R., Targeting tumor vasculature: Reality or a
dream?.J. Drug Targeting, 10:529-533 (2002). .
Duncan, R. et al., Polymers containing enzymatically degradable
bonds, 7. Design of oligopeptide side chains in poly
[N-(2-hydroxypropyl)methacrylamide] copolymers to promote efficient
degradation by lysosomal enzymes, Makromol. Chem., 184:1997-2008
(1983). .
Foekens, J.A. et al., Prognostic significance of cathepsins B and L
in primary human breast cancer.J. Clinical Oncol., 16:1013-1021
(1998). .
Gianasi, E. et al., HPMA copolymer platinates as novel antitumour
agents: in vitro properties, pharmacokinetics and antitumour
activity in vivo, Eur. J. Cancer, 35:994-1002 (1999). .
Kusaka, M. et al. Cytostatic inhibition of endothelial cell growth
by the angiogenesis inhibitor TNP-470 (AGM-1470), Br. J. Cancer.
69:212-216 (1994). .
Greene, A.K. et al., Endothelial-directed hepatic regeneration
after partial hepatectomy, Ann. Surg., 237:530-535 (2003). .
Drixler, T.A. et al., Liver regeneration is an angiogenesis-
associated phenomenon, Ann. Surg., 236:703-712 (2002). .
Klein, S.A. et al., Angiogenesis inhibitor TNP-470 inhibits murine
cutaneous wound healing, J. Surg. Res., 82:268-274 (1999). .
Whalen, C.T. et al., Assay of TNP-470 and its two major metabolites
in human plasma by high-performance liquid chromatography-mass
spectrometry, J. Chromatographic Sci., 40:214-218 (2002). .
Brocchini, S. et al., Polymer-Drug conjugates: drug release from
pendent linkers. in Encyclopaedia of controlled release (ed.
Mathiovitz, E.) 786-816 (New York: Wiley, 1999). .
Duncan, R. et al., Polymer-drug conjugates, PDEPT and PELT: basic
principles for design and transfer from the laboratory to clinic,
J. Controlled Release, 74:135-146 (2001). .
Vasey, P.A. et al., Phase I clinical and pharmacokinetic study of
PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]:
first member of a new class of chemotherapeutic agents-drug-polymer
conjugates, Cancer Research Campaign Phase I/II Committee, Clinical
Cancer Res., 5:83-94 (1999). .
Seymour, L.W. et al., Tumour tropism and anti-cancer efficacy of
polymer-based doxorubicin prodrugs in the treatment of subcutaneous
murine B16F10 melanoma, Br. J. Cancer, 70:636-641 (1994). .
Dvorak, H.F. et al., Identification and characterization of the
blood vessels of solid tumors that are leaky to circulating
macromolecules. Am. J. Pathology, 133:95-109 (1988). .
Griffith, E.C. et al., Methionine aminopeptidase (type 2) is the
common target for angiogenesis inhibitors AGM-1470 and ovalicin,
Chem. and Biol., 4, 461-471 (1997). .
Auerbach, R. et al., Angiogenesis assays: problems and pitfalls,
Cancer Metastasis Rev., 19:167-172 (2000). .
Seymour, L.W. et al., Hepatic drug targeting: phase I evaluation of
polymer-bound doxorubicin., J. Clinical Oncol., 20:1668-1676
(2002). .
Francis, G.E. et al., PEG-modified proteins. in Stability of
Proteins Pharmaceuticals (Part B) (ed. Ahem Tj, M.M.) 235-263
(Plenum Press, New York, 1992). .
Ho, D.H. et al., Clinical pharmacology of polyethylene
glycol-L-asparaginase, Drug Metabolism Disposition, 14:349-352
(1986). .
O'Reilly, M.S. et al., Angiostatin: a novel angiogenesis inhibitor
that mediates the suppression of metastases by a Lewis lung
carcinoma, Cell, 79:315-328 (1994). .
Folkman, J. et al., Long-term culture of capillary endothelial
cells, Proc. Natl. Acad. Sci. USA, 76:5217-5221 (1979). .
Waynforth, H.B. Routes and methods of administration, Intracerebral
injection. in Experimental and Surgical technique in the rat, vol.
2.9 34-36 (Academic Press, London, 1980). .
Seymour, L.W. et al., The pharmacokinetics of polymer-bound
adriamycin, Biochemical Pharmacology, 39:1125-1131 (1990). .
Yeh, J.R. et al., The antiangiogenic agent TNP-470 requires p53 and
p21.sup.CIP/WAF for endothelial cell growth arrest, Proc. Natl.
Acad. Sc.i USA, 97:12782-12787 (2000). .
Zhang, Y. et al., Cell cycle inhibition by the anti-angiogenic
agent TNP-470 is mediated by p53 and p21.sup.WAF1/CIP1, Proc. Natl.
Acad. Sci. USA, 97:6427-6432 (2000). .
Seymour, L.W. et al., N-(2-hydroxypropyl) methacrylamide copolymers
targeted to the hepatocyte galactose-receptor: pharmacokinetics in
DBA.sub.2 mice, Br. J. Cancer, 63:859-866 (1991). .
Folkman, J. Tumor angiogenesis. in Accomplishments in cancer
research (eds. Wells, S.J. & Sharp, P.) 32-44 (Lippincott
Williams & Wilkins, New York, 1998)..
|
Primary Examiner: Balasubramanian; Venkataraman
Attorney, Agent or Firm: Nixon Peabody LLP
Parent Case Text
CROSS-REFERENCE
This application is a Continuation-in-Part of International
Application No. PCT/US03/10976 filed on Apr. 10, 2003, designating
the United States, which claims benefit under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Application Nos. 60/371,791 filed Apr.
11, 2002 and 60/414,705 filed on Sep. 30, 2002.
Claims
What is claimed is:
1. A compound of the formula ##STR7##
wherein R is (CH.sub.2).sub.n R', where n is 1 to 3, R' is
NH.sub.2, OH or SH, or a pharmaceutically acceptable salt
thereof.
2. The compound of claim 1, wherein R' is (CH.sub.2).sub.2
NH.sub.2.
3. A method of inhibiting angiogenesis in a mammal having undesired
angiogenesis comprising administering an effective amount of the
compound of claim 1.
Description
BACKGROUND OF THE INVENTION
In recent years, it has become clear that angiogenesis, the growth
of new capillary blood vessels from pre-existing vasculature, is
important not only in physiological processes such as embryonic
development, the female reproductive cycle, wound healing, and
organ and tissue regeneration, but also in pathological processes
such as tumor progression and metastasis.sup.1. Angiogenesis is now
recognized as a critical process for all malignancies.sup.2,3. As a
result, the microvascular endothelial cell, which is recruited by
tumors, has become an important second target in cancer therapy. It
is widely accepted that the endothelial cell target, unlike the
tumor cells themselves is genetically stable.sup.1. Antiangiogenic
agents have recently emerged as a new class of drugs; however, the
optimal means to use these agents alone or in combination with drug
delivery systems and with conventional chemotherapy have not yet
been fully elucidated.
The hypothesis that tumor growth is angiogenesis-dependent is
supported by biological and pharmacological evidence.sup.4 and
confirmed by genetic evidence.sup.3,5-7. Both types of evidence
provide a scientific basis for current clinical trials of
angiogenesis inhibitors. Increased tumor angiogenesis.sup.4,8 and
elevated levels of proangiogenic factors such as vascular
endothelial growth factor (VEGF/VPF).sup.8,9, basic fibroblast
growth factor (bFGF).sup.8, and interleukin-8 (IL-8).sup.10
correlate with decreased survival and increased risk of relapse in
studies of patients with malignant solid tumors. The importance of
angiogenesis is further supported by the observation that
antiangiogenic agents inhibit tumor growth in a variety of animal
models.
In the U.S. there are currently more than 30 angiogenesis
inhibitors in various clinical trials for late-stage cancer. One of
these angiogenesis inhibitors, O-(chloracetyl-carbamoyl) fumagillol
(TNP-470), is a low molecular weight synthetic analogue of
fumagillin.sup.11, a compound secreted by the fungus Aspergillus
fumigatus fresenius. TNP-470 is a potent endothelial inhibitor in
vitro.sup.12. Recently, TNP-470 has been tested as a potential new
anticancer agent. In animal models, TNP-470 has the broadest
anticancer spectrum of any known agent.sup.4,13. TNP-470 inhibited
the growth of murine tumors up to 91%, human tumors up to 100% and
metastatic tumors up to 100% in mice (reviewed in ref. .sup.13) In
most studies, mice were treated at the same optimal dose of 30
mg/kg subcutaneously every other day. In clinical trials TNP-470
has shown evidence of antitumor activity when used as a single
agent, with a number of objective responses reported with relapsed
and refractory malignancies.sup.14-16. It has also shown promise
when used in combination with conventional chemotherapy.sup.17,18.
However, many patients experience neurotoxicity (malaise, rare
seizures, asthenia, anxiety and dysphoria).sup.16,17,19,20 at doses
where antitumor activity has been seen. Because of dose-limiting
neurotoxicity, TNP-470 has been tested using multiple dosing
regimens, but these attempts to limit its toxicity have been
unsuccessful. With few exceptions, weight loss or failure to gain
weight was observed in animals receiving TNP-470.sup.21, and two
reports noted a decrease in splenic weight.sup.22,23. Therefore,
modifications of TNP-470 that can retain or increase its activity
while reducing its toxicity are highly desirable.
SUMMARY OF THE INVENTION
The present invention relates to conjugates of water-soluble
polymers and o-(chloracetyl-carbamoyl) fumagillol (TNP-470) and use
of those conjugates as specific intracellular carriers of the
TNP-470 into tumor vessels. This invention also relates to an
intermediate formed in the synthesis of these conjugates and its
use. The present invention further relates to use of those
conjugates to lower the neurotoxicity of TNP-470. Preferably, the
polymer has a molecular weight in the range of 100 Da to 800 kDa.
More preferably, the polymer has a molecular weight no greater than
60 kDa. Most preferably, the polymer has a molecular weight in the
range of 15 kDa to 40 kDa.
Preferred polymers are HPMA copolymers. HPMA copolymers are
biocompatible, non-immunogenic and non-toxic carriers that enable
specific delivery into tumor endothelial cells overcoming
limitations of drug-related toxicities (Duncan, et al., Hum Exp
Toxicol, 17:93-104 (1998)). Moreover, their body distribution is
well characterized and they are known to accumulate selectively in
the tumor site due to the enhanced permeability and retention (EPR)
effect (Maeda, et al., J Controlled Release, 65:271-284 (2000)).
The conjugate can also include a targeting moiety to direct the
conjugate to sites of endothelial cell proliferation or cancer
cells or to specific receptors or markers associated with
proliferating endothelial cells.
TNP-470 can be conjugated to a polymer via nucleophelic attack on
the .alpha.-carboxyl releasing the chlorine. The intermediate
formed has the pertinent structure, ##STR1##
wherein R is (CH.sub.2).sub.n R', where n is 0 to 3, R' is
NH.sub.2, O or S.
The TNP-470 conjugate when cleaved enzymatically forms the above
described structure wherein R is (CH.sub.2).sub.2 NH.sub.2.
This intermediate, or a pharmaceutically acceptable salt thereof,
can also be used as an anti-tumor agent. It, like the polymer
conjugate, is antiangiogenic and would also form the active TNP-470
metabolite.
The data presented herein demonstrate that, for example, TNP-470
conjugated to an HPMA copolymer: (i) avoid high peak drug levels in
the circulation (ii) avoid penetration of TNP-470 to the
cerebrospinal fluid and thus prevent the problem of neurotoxicity;
(iii) prolong its half-life; (iv) facilitate the accumulation of
TNP-470 in tissues involving neovascularization; (v) convert
TNP-470 to a highly effective and widely useful angiogenesis
inhibitor. We have also surprisingly discovered that conjugating
TNP-470 to HPMA results in a water soluble composition.
The present invention further relates to use of the conjugates in
methods of treating angiogenic diseases and decreasing
neurotoxicity of TNP-470. Angiogenic disease amenable to treatment
with the present invention include but are not limited to diabetic
retinopathy, macular degeneration, retrolental fibroplasia,
trachoma, neovascular glaucoma, psoriases, angio-fibromas, immune
and non-immune inflammation, capillary formation within
atherosclerotic plaques, hemangiomas, excessive wound repair, solid
tumors, metastases, Kaposi's sarcoma and the like.
In accordance with the present invention, if polymer a having a
molecular weight greater than 60 kDa is used, it is preferred that
the polymer be a degradable polymer or inert. As used herein, a
"degradable" polymer is one that breaks down in vivo to components
having a molecular weight no greater than 60 kD. As defined herein,
poly vinyl alcohol (PVA) is not a degradable polymer.
Other aspects of the invention are disclosed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A illustrates the structure of HPMA copolymer-Gly-Phe-Leu-Gly
(SEQ ID NO: 1)-ethylenediamine-TNP-470. FIG. 1B shows in vitro
release of TNP-470 from HPMA copolymer in the presence
(-.box-solid.-) and absence ((-.diamond-solid.-) of cathepsin
B.
FIG. 2A shows inhibition of BCE proliferation in vitro after 72 h.
TNP470 (-.tangle-solidup.-) and HPMA copolymer-Gly-Phe-Leu-Gly (SEQ
ID NO: 1)-en-TNP-470 (-.box-solid.-) had similar cytostatic effect
on bFGF-induced proliferation of endothelial cells at doses lower
than 1 .mu.g/ml and cytotoxic effect at doses higher than 1
.mu.g/ml. The dotted line represents the proliferation of
bFGF-induced BCE cells ( - - - ) and the solid line represents the
BCE cell proliferation in the absence of bFGF (-). FIG. 2B shows
the chick aortic ring endothelial sprouting assay. The effect of
TNP-470 (central panel) and HPMA copolymer-Gly-Phe-Leu-Gly (SEQ ID
NO: 1)-en-TNP-470 (right panel) at 100 pg/ml TNP-470
equivalent-dose are shown; and a control chick aortic ring (left
panel) with abundant sprouting.
FIG. 3A shows a schematic representation of the hepatectomy model.
Untreated livers regenerate in 8 days, but they do not regenerate
when treated with TNP-470 30 mg/kg/q.o.d s.c. FIG. 3B shows that
free TNP-470 (stripes columns) inhibited liver regeneration when
used at 30 mg/kg/q.o.d s.c. However, it did not inhibit liver
regeneration at other dosing schedules. Conjugated TNP-470 (solid
columns) inhibited liver regeneration at 30 mg/kg/q.o.d s.c. or 60
mg/kg/q.2.d s.c. or even at a single dose of 120 mg/kg/day of
operation s.c. compared to the control regenerated group (dotted
columns). FIG. 3C shows that free TNP-470 (-.circle-solid.-) causes
delay in newborn mice development, but did not affect body weight
when used in the conjugated form (-.tangle-solidup.-) similar to
the control mice (-.box-solid.-). Arrows represent days of
treatment. Data represent mean.+-.SE, n=9 mice per group.
FIG. 4 shows antitumour activity measured using male SCID mice
bearing A2058 human melanoma. FIG. 4A shows the effect of TNP-470
(-.circle-solid.-); HPMA copolymer-Gly-Phe-Leu-Gly (SEQ ID NO:
1)-en-TNP-470 (-.tangle-solidup.-); and control mice
(-.box-solid.-) on tumors. Data represent mean.+-.SE, n=8 mice per
group. P values of <0.05 were marked as *, P<0.03 **,
P<0.01 ***. FIG. 4B shows SCID mice and excised tumors
correlating to panel (A) at day 8 of treatment. FIG. 4C shows H
& E staining of tumors excised from animals in different groups
on day 8 at high and low power.
FIG. 5 shows antitumour activity measured using male C57 mice
bearing LLC. FIG. 5A shows the effect of TNP-470 at 30 mg/kg/q.o.d.
s.c. (-.circle-solid.-); HPMA copolymer-Gly-Phe-Leu-Gly (SEQ ID NO:
1)-en-TNP-470 at 30 mg/kg/q.o.d. s.c. (-.tangle-solidup.-) on tumor
growth; control mice (-.box-solid.-) are also shown. Data represent
mean.+-.SE, n=10 mice per group. FIG. 5B shows representative C57
mice correlating to (A) on day 10 following treatment. FIG. 5C
shows dose escalation of EPMA copolymer-Gly-Phe-Leu-Gly (SEQ ID NO:
1)-en-TNP-470: at 30 (-.tangle-solidup.-), at 60 (-.circle-solid.-)
and at 90 mg/kg/q.o.d. (-.diamond-solid.-) and control mice
(-.box-solid.-) are shown. Data are mean.+-.SE, n=10 mice per
group. FIG. 5D shows C57 mice correlaing to (C). P values of
<0.05 were marked as *, P<0.03 as **, P<0.01 as ***.
FIG. 6 shows the results of a Miles assay.
FIG. 7 shows the effect of TNP-470 on serum-induced cell
proliferation. Inhibition of BCE (open symbols) and A2058 (closed
symbols) cell proliferation in vitro after 72 h. TNP-470
(-.circle-solid.-) and HPMA copolymer-GFLG (SEQ ID NO:
1)-en-TNP-470 (-.tangle-solidup.-) had similar cytostatic effect on
bFGF-induced proliferation of endothelial cells at doses lower than
1 .mu.g/ml and cytotoxic effect at doses higher than 1 .mu.g/ml.
The dotted line represents the proliferation of bFGF-induced BCE or
serum-induced A2058 cells (--) and the solid line represents the
BCE and A2058 cell proliferation in the absence of bFGF or serum,
respectively ( - - - ).
FIG. 8 shows HPMA copolymer-TNP-470 accumulation in tumors and
serum. Panel 8(a) TNP-470 species extracted from tumors. Panel 8(b)
TNP-470 extracted from serum. Free TNP-470 concentration was
negligible at these time points. Values are mean.+-.S.E., n-3 mice
per group.
FIG. 9 shows the effects of TNP-470 and HPMA copolymer-TNP-470 on
the motor skills of mice using the rotorod test. Panel 9(a) Mouse
on a rotorod treadmill. Panel 9(b) Mice were treated with free
TNP-470 (30 mg/kg q.o.d. s.c.; green columns), HPMA
copolymer-TNP-470 (30 mg/kg q.o.d. s.c.; red columns), or saline
(250 .mu.l q.o.d. s.c.; blue columns) for 5 weeks. The mean time
each group remained on the rotating rod is shown in the figure.
Data are mean.+-.S.E. n-5 mice per group. Panel 9(c) Body weight of
mice treated with free TNP-470 (30 mg/kg q.o.d. s.c.)
(-.tangle-solidup.-), HPMA copolymer-TNP-470 (30 mg/kg q.o.d. s.c.)
(-.cndot.-), or saline (250 .mu.l q.o.d. s.c.) (-.box-solid.-) for
5 weeks.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to polymer and copolymer conjugates
of TNP-470 and TNP-470 species.
In accordance with the present invention, the TNP-470 is linked to
a water soluble degradable or non-degradable polymer having a
molecular weight in the range of 100 Da to 800 kDa. The components
of the polymeric backbone may comprise acrylic polymers, alkene
polymers, urethanepolymers, amide polymers, polyimines,
polysaccharides and ester polymers. Preferably the polymer is
synthetic rather than being a natural polymer or derivative
thereof. Preferably the backbone components comprise derivatised
polyethyleneglycol and poly(hydroxyalkyl(alk)acrylamide), most
preferably amine derivatised polyethyleneglycol or
hydroxypropyl(meth)acrylamide-methacrylic acid copolymer or
derivative thereof. Dextran/dextrin and polyethylene glycol
polymers, or derivatives thereof, may also be used. Preferably, the
polymer has a molecular weight no greater than 60 kDa. A most
preferred molecular weight range is 15 to 40 kDa.
The TNP-470 and the polymer are conjugated by use of a linker,
preferably a cleavable peptide linkage. Most preferably, the
peptide linkage is capable of being cleaved by preselected cellular
enzymes, for instance, those found in lysosomes of cancerous cells
or proliferating endothelial cells. Alternatively, an acid
hydrolysable linker could comprise an ester or amide linkage and be
for instance, a cis-aconityl linkage. A pH sensitive linker may
also be used.
Cleavage of the linker of the conjugate results in release of
active TNP-470. Thus the TNP-470 must be conjugated with the
polymer in a way that does not alter the activity of the agent. The
linker preferably comprises at least one cleavable peptide bond.
Preferably the linker is an enzyme cleavable oligopeptide group
preferably comprising sufficient amino acid units to allow specific
binding and cleavage by a selected cellular enzyme. Preferably the
linker is at least two amino acids long, more preferably at least
three amino acids long. For example; TNP470 can be conjugated to
HPMA copolymer-Gly-Phe-Leu-Gly (SEQ ID NO: 1)-ethylendiamine via
nucleophilic attack on the .alpha.-carbonyl on the TNP-470
releasing the chlorine to form a compound of formula 1,
##STR2##
wherein R is (CH.sub.2).sub.n R', where n is 0 to 3, preferably n
is 2, and R' is NH.sub.2, O or S. For instance, HPMA
copolymer-Gly-Phe-Leu-Gly (SEQ ID NO: 1)-ethylendiamine (100 mg)
can be dissolved in DMF (1.0 ml). Then, TNP-470 (100 mg) can be
dissolved in 1.0 ml DMF and added to the solution. The mixture is
stirred in the dark at 4.degree. C. for 12 h. DMF is then
evaporated and the product, HPMA copolymer-TNP-470 conjugate is
redissolved in water, dialyzed (10 kDa MWCO) against water to
exclude free TNP-470 and other low molecular weight contaminants,
lyophilized and stored at -20.degree. C. Reverse phase HPLC
analysis using a C18 column, is used to characterize the conjugate.
This conjugated structure can be cleaved enzymatically between the
glycine residue of the peptide and the ethylenediamine residue (See
FIG. 1A).
The resultant product is 6-O-(N-ethylaminoglycinylcarbmoyl)
fumagillol, which has the structure shown below. ##STR3##
This is the compound of formula 1, where R is --(CH.sub.2).sub.2
NH.sub.2. This compound has a bis-epoxide functionality.
Accordingly, it will also have anti-tumor activity, particularly
antiangiogenic activity. This compound, or its' pharmaceutically
acceptable salt, should be able to be cleaved, like TNP-470, to the
active metabolite set forth below (2). ##STR4##
This should be water soluble. This product can be used by itself
without the conjugate. The compound can be modified by known means
and should still retain its water soluble, as well as its
antiangiogenic, properties. These modifications can be made by
known means, such as those used with other fumagillian derivatives.
Preferably nucleophiles of the formula (NH.sub.2).sub.n R, wherein
n is 1 to 2 and R is H, O or S can be used to substitute for the Cl
of TNP-470.
One mode for synthesis of the compound of formula 1, where R is
--(CH.sub.2).sub.2 NH.sub.2 is illustrated below. ##STR5##
Also included within the scope of the present invention are
compositions that comprise, as an active ingredient, the organic
and inorganic addition salts of the above-described compound and
combinations thereof; optionally, in association with a conjugate,
diluent, slow release matrix, or coating.
The organic or inorganic addition salts of the water soluble
antiangiogenic compounds and conjugates thereof contemplated to be
within the scope of the present invention include salts of such
organic moieties as acetate, trifluoroacetate, oxalate, valerate,
oleate, laurate, benzoate, lactate, tosylate, citrate, maleate,
fumarate, succinate, tartrate, naphthalate, and the like; and such
inorganic moieties as Group I (i.e., alkali metal salts), Group II
(i.e. alkaline earth metal salts) ammonium and protamine salts,
zinc, iron, and the like with counterions such as chloride,
bromide, sulfate, phosphate and the like, as well as the organic
moieties referred to above.
Pharmaceutically acceptable salts are preferred when administration
to human subjects is contemplated. Such salts include the non-toxic
alkali metal, alkaline earth metal and ammonium salts commonly used
in the pharmaceutical industry including sodium, potassium,
lithium, calcium, magnesium, barium, ammonium and protamine salts
which are prepared by methods well known in the art. The term also
includes non-toxic acid addition salts which are generally prepared
by reacting the compounds of this invention with a suitable organic
or inorganic acid. Representative salts include hydrochloride,
hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate,
oleate, laurate, borate, benzoate, lactate, phosphate, tosylate,
citrate, maleate, fumarate, succinate, tartrate, napthylate and the
like.
Preferred polymers for use with the present invention are HPMA
copolymers with methacrylic acid with pendent oligopeptide groups
joined via peptide bonds to the methacrylic acid with activated
carboxylic terminal groups such as paranitrophenyl derivatives or
ethylene diamine.
In a preferred embodiment the polymeric backbone comprises a
hydroxyalkyl(alk)acrylamide methacrylamide copolymer, most
preferably a copolymer of hydroxypropyl(meth)acrylamide copolymer
(HPMA). The HPMA prior to attachment of the TNP-470 has the
structure set forth below: ##STR6##
y can be in the range of 0.01-100 and x can be in the range
0-99.99. y is preferably in the range of 0.04-20 and x is
preferably in the range 80-99.96. Preferably L is an oligopeptide
group containing between 2 and 10 peptide moieties, most preferably
3 or 4.
In a most preferred embodiment, L is a Gly-Phe-Leu-Gly-(SEQ ID NO:
1) linkage. In one embodiment, U is an ONp group, wherein Np is a
p-nitrophenyl group. Preferably y is in the range 0.3 to 15 and x
is in the range of 99.7 to 85. Most preferably, y is in the range
of 5-10 and x is in the range of 90-95. In a more preferred
embodiment, the polymeric backbone is HPMA
copolymer-Gly-Phe-Leu-Gly (SEQ ID NO: 1)-ethylenediamine having the
values for x and y as defined above.
In a most preferred embodiment of HPMA copolymer TNP-470 conjugate
has the structure set forth in FIG. 1A.
HPMA polymers and use thereof are disclosed in WO 01/36002.
In another embodiment, the conjugate is a liposome/TNP-470
conjugate. Preferably, the conjugate is a pegylated liposomal
TNP-470. An exemplary conjugate comprises: a) TNP-470; b)
N-(carbonyl-methoxypolyethylene glycol
2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt;
c) fully hydrogenated soy phosphatidylcholine; d) cholesterol;
Histidine, hydrochloric acid and/or sodium hydroxide, ammonium
sulfate, and sucrose; wherein the weight percentage ratio of
a:b:c:d is about 1.0:1.60:4.80:1.60 mg/mL respectively.
While the antiangiogenic agent conjugate and/or water soluble
antiangiogenic compound may rely for its localization at a solid
tumor, or other sites of active angiogenesis, primarily upon EPR,
it may be desirable to attach ligands allowing active targeting. A
preferred targeting ligand is directed to the integrin a V.beta.3
and contains the tripeptide sequence RGD or RGD4C (cyclic RGD).
Antibodies or ligands directed to cell receptors or other
upregulated molecules present on the cell surface may also be used.
See, e.g. 28.
The conjugate and the water soluble antiangiogenic compound of the
present invention is useful in inhibiting the angiogenic function
of endothelial cells both in vitro and in vivo. Of particular
interest is the prevention or inhibition of endothelial cell
differentiation into capillary structures. The endothelial cells
amenable to inhibition by the conjugate are present at several
sites in a mammal and include but are not limited to dermis,
epidermis, endometrium, retina, surgical sites, gastrointestinal
tract, liver, kidney, reproductive system, skin, bone, muscle,
endocrine system, brain, lymphoid system, central nervous system,
respiratory system, umbilical cord, breast tissue, urinary tract
and the like. The method of treatment of the present invention
using the conjugate and compound is particularly useful in
preventing or inhibiting angiogenesis by endothelial cells at sites
of inflammation and tumorigenesis.
The conjugate and compound is particularly useful in methods of
inhibiting angiogenesis at a site of tumorigenesis in a mammal. The
conjugate and compound administered at such sites prevents or
inhibits blood vessel formation at the site thereby inhibiting the
development and growth of the tumor. Tumors which may be prevented
or inhibited by preventing or inhibiting angiogenesis with the
conjugate include but are not limited to melanoma, metastases,
adenocarcinoma, sarcomas, thymoma, lymphoma, lung tumors, liver
tumors, colon tumors, kidney tumors, non-Hodgkins lymphoma,
Hodgkins lymphoma, leukemias, uterine tumors, breast tumors,
prostate tumors, renal tumors, ovarian tumors, pancreatic tumors,
brain tumors, testicular tumors, bone tumors, muscle tumors, tumors
of the placenta, gastric tumors and the like.
In providing a mammal with the conjugate and/or compound,
preferably a human, the dosage of administered conjugate will vary
depending upon such factors as the mammal's age, weight, height,
sex, general medical condition, previous medical history, disease
progression, tumor burden, route of administration, formulation and
the like. For example, a suitable dose of the conjugate or compound
for a mammal in need of treatment as described herein is in the
range of about 1 mg to about 2000 mg TNP-470 per kilogram of body
weight.
The route of administration may be intravenous (I.V.),
intramuscular (I.M.), subcutaneous (S.C.), intradermal (I.D.),
intraperitoneal (I.P.), intrathecal (I.T.), intrapleural,
intrauterine, rectal, vaginal, topical, intratumor and the
like.
The present invention encompasses combination therapy in which the
conjugate or compound is used in combination with a
chemotherapeutic agent such as Taxol, cyclophosphamide, cisplatin,
gancyclovir and the like. The chemotherapeutic agent may also be
conjugated to a polymer. Such a therapy is particularly useful in
situations in which the mammal to be treated has a large
preexisting tumor mass which is well vascularized. The
chemotherapeutic agent serves to reduce the tumor mass and the
conjugate prevents or inhibits neovascularization within or
surrounding the tumor mass. The chemotherapeutic agent may also be
administered at lower doses than normally used and at such doses
may act as an antiangiogenic agent.
The present invention is further illustrated by the following
Examples. These examples are provided to aid in the understanding
of the invention and are not construed as a limitation thereof.
EXAMPLE 1
Methods
Materials
A random copolymer of HPMA copolymerized with
methacryloyl-Gly-Phe-Leu-Gly (SEQ ID NO: 1)-p-nitrophenyl ester
(HPMA copolymer-MA-GFLG (SEQ ID NO: 1)-ONp) incorporating
approximately 10 mol % of the MA-GFLG (SEQ ID NO: 1)-ONp monomer
units was prepared as previously reported .sup.24 provided by
Polymer Laboratories (UK). The polymeric precursor was used for
ethylenediamine (en) incorporation and the product HPMA
copolymer-GFLG (SEQ ID NO: 1)-en had a Mw of 31,600 Da and
polydispersity (PD) of 1.66. TNP-470 was kindly provided by Douglas
Figg from the NCI (USA). 2-Propanol, methanol, orthophosphoric acid
and chloroform were from Sigma (all HPLC grade). Dimethylformamide
(DMF) and dimethylsulfoxide (DMSO) were from Aldrich (USA). All
other chemicals were of analytical grade from Aldrich (USA) and
Fisher Chemicals (USA) unless otherwise stated. Vivacell 70 ml (10
kDa MW cut-off PES) was from VivaScience (USA). Isoflurane was
purchased from Baxter Healthcare Corporation (USA). Matrigel
basement membrane matrix (from Engeibreth-Hoim-Swarm mouse tumor)
was purchased from Becton Dickinson (USA). Avertin was purchased
from Fisher (USA).
A2058 human melanoma cells were from the ATCC. LLC cells were
passaged from mouse to mouse as previously described.sup.47. Cells
were maintained in DMEM medium containing 10% inactivated fetal
bovine serum (Life Technologies, Inc.), 0.29 mg/ml L-glutamine, 100
units/ml penicillin and 100 .mu.g/ml streptomycin (GPS) (Gibco) in
a humidified 5% CO.sub.2 incubator at 37.degree. C. BCE cells were
isolated in our laboratory, and cultured in a humidified 10%
CO.sub.2 incubator at 37.degree. C. as described.sup.48. BCE cells
were grown in DMEM medium supplemented with 10% bovine calf serum
(BCS), GPS, and 3 ng/ml basic fibroblast growth factor (bFGF).
C57BL/6J mice were purchased from Jackson Laboratories (USA), SCID
mice were from Massachusetts General Hospital (USA) and BALB/c mice
were from Charles River (USA).
Synthesis
TNP-470 was conjugated to HPMA copolymer-Gly-Phe-Leu-Gly (SEQ ID
NO: 1)-ethylendiamine via nucleophilic attack on the
.alpha.-carbonyl on the TNP470 releasing the chlorine. HPMA
copolymer-Gly-Phe-Leu-Gly (SEQ ID NO: 1)-ethylendiamine (100 mg)
was dissolved in DMF (1.0 ml). Then, TNP-470 (100 mg) was dissolved
in 1.0 ml DMF and added to the solution. The mixture was stirred in
the dark at 4.degree. C. for 12 h. DMF was evaporated and the
product, HPMA copolymer-TNP-470 conjugate was redissolved in water,
dialyzed (10 kDa MWCO) against water to exclude free TNP-470 and
other low molecular weight contaminants, lyophilized and stored at
-20.degree. C. Reverse phase HPLC analysis using a C18 column, was
used to characterize the conjugate.
Bovine Capillary Endothelial (BCE) Cell Proliferation Assay
BCE cells were obtained and grown as previously described.sup.48.
For the proliferation assay, cells were washed with PBS and
dispersed in a 0.05% trypsin solution. Cells were suspended (15,000
cells/ml) in DMEM supplemented with 10% BCS and 1% GPS, plated onto
gelatinized 24-well culture plates (0.5 ml/well), and incubated for
24 h (37.degree. C., 10% CO.sub.2). The media was replaced with
0.25 ml of DMEM, 5% BCS and 1% GPS and the test sample applied.
Cells were challenged with free or conjugated TNP-470 (10 pg/ml to
1 .mu.g/ml TNP-470-equivalent concentration). After 30 min of
incubation, media and bFGF were added to obtain a final volume of
0.5 ml of DMEM, 5% BCS, 1% GPS and 1 ng/ml bFGF. Control cells were
grown with or without bFGF. After 72 hr, cells were dispersed in
trypsin, resuspended in Hematall (Fisher Scientific, Pittsburgh,
Pa.), and counted in a Coulter counter.
Chick Aortic Ring Assay:
Aortic arches were dissected from day-14 chick embryos, cut into
cross-sectional fragments, and implanted in vitro in Matrigel using
a modification of methods previously described (V. Muthukkaruppan,
personal communication). When cultured in MCDB-131 medium
supplemented with 5% fetal bovine serum, endothelial cells sprouted
and vascular channel formation occurred within 24-48 hours. Free or
conjugated TNP-470 (10 pg/ml to 1 .mu.g/ml) was added to the
culture.
Hepatectomy Model
Male C57BL/6J mice underwent a partial hepatectomy through a
midline incision after general anesthesia with isoflourane.sup.33.
Free or conjugated TNP-470 (30 mg/kg) were given s.c. every other
day for 8 days beginning on the day of surgery according to the
scheme described in FIG. 4a. Alternatively, the doses given were 60
mg/kg the day of surgery and 4 days later or 120 mg/kg once on the
day of the partial hepatectomy. The liver was harvested on the
8.sup.th day, weighed and analyzed by histology.
Evaluation of the Body Distribution of Free TNP-470 and HPMA
Copolymer-TNP-470 in Mice Bearing s.c. LLC
Male C57BL/6J mice were inoculated with 5.times.10.sup.6 viable LLC
cells s.c. and the tumor was allowed to grow to a volume of
approximately 100 mm.sup.3. Animals were injected i.v. with free or
conjugated TNP-470 (30 mg/kg). Intracerebral withdrawal of CSF from
the brain of C57BL/6J mice was performed using a Model 310
stereotaxic apparatus (Stoelting Co., Wooddale Ill.) according to
stereotaxic coordinates described in the mouse brain atlas.sup.49
and the method described in Waynforth.sup.50. Once the desired
amount of fluid was obtained (approximately 20 .mu.l), the animal
was euthanized via cervical dislocation at times up to 72 h.
Tumors, major organs, blood, urine and CSF were collected and
homogenized. Then a TNP-470 species (sometimes referred to herein
as TNP-470) was extracted in chloroform. Following evaporation of
the chloroform, samples were redissolved and high-performance
liquid chromatography (HPLC)/tandem Mass Spectrometry (LC-MS/MS)
was used to determine the amount of free TNP-470 in the samples as
previously described.sup.36.
Evaluation of Antitumor Activity of HPMA Copolymer-TNP-470
Male C57BL/6J mice (.about.8 weeks, .about.20 g) were inoculated
with 5.times.10.sup.6 viable LLC or A2058 melanoma cells s.c. The
tumors were allowed to grow to a volume of approximately 100
mm.sup.3. Animals were injected i.v. with free TNP-470 or HPMA
copolymer-TNP-470 (30 mg/Kg TNP-equiv.) or saline (250 .mu.l i.v.).
Each group consisted of 5 mice. Mice were euthanized when tumors
reached or surpassed a size equivalent to 30% of their body weight.
Animals were weighed daily and observed for signs of tumor
progression and euthanized if their body weight decreased below 80%
of their starting weight. Animals were monitored for general
health, weight loss, and tumor progression. At termination, mice
underwent post-mortem examination and tumors were dissected and
weighed. A similar experiment was repeated in which treatment with
escalating doses of the conjugate was initiated when tumors
reached. 500 mm.sup.3. The same dosing schedule was repeated with
white SCID male mice (.about.8 weeks, 20 g) inoculated with
5.times.10.sup.6 viable A2058 human melanoma cells s.c. and treated
as described above.
Statistical Methods
All of the in vitro data are expressed as the mean.+-.standard
deviation of the mean (S.D.). All of the in vivo data are expressed
as the mean.+-.standard error of the mean (S.E.). Statistical
significance was assessed using the Student's t-test. P values of
0.05 or less were considered statistically significant.
Results
Synthesis and Characterization
HPMA copolymer-Gly-Phe-Leu-Gly (SEQ ID NO:
1)-ethylenediamine-TNP-470 conjugate (FIG. 1A) was synthesized,
purified and characterized by HPLC. Gly-Phe-Leu-Gly (SEQ ID NO: 1)
polymer-TNP-470 linker was designed to permit intralysosomal TNP470
liberation due to action of the lysosomal cysteine
proteases.sup.29, such as cathepsin B. It has been shown that
cathepsin B is overexpressed in many tumor cells.sup.30. The
conjugate accumulates selectively in the tumor tissue due to the
EPR effect and is slowly internalized into endothelial cells in the
tumor bed by fluid-phase pinocytosis. The conjugate should not
internalize into normal quiescent endothelial cells, hence will not
be exposed to lysosomal enzymes leaving the linker intact. Free
TNP-470 eluted as a single peak with a retention time of 13.0 mm
while the conjugate eluted as a wider peak at 10.0 mm (results not
shown). Free drug was negligible (<0.01% of total TNP-470)
following repeated purification by dialysis. TNP-470 is not
water-soluble but became soluble following conjugation with HPMA
copolymer. The conjugate was stable for three days in phosphate
buffered saline or citrate buffer, pH 5.5, 0.2 M at 37.degree. C.
However, under the same conditions with the addition of the
lysosomal enzyme cathepsin B, the linker between the polymer and
the drug (Gly-Phe-Leu-Gly.sup.31) (SEQ ID NO: 1) was cleaved and
TNP-470 was released (FIG. 1B). These conditions imitate the
lysosomal environment in endothelial cells where lysosomal enzymes,
such as cathepsin B, are present. TNP-470 release from the
conjugate reached a plateau within 5 h of incubation with cathepsin
B and did not increase appreciably even after 5 days. The incubated
solution was then analyzed and had a TNP-470 content of
approximately 10 mol %. We next tested the HPMA copolymer-TNP-470
conjugate activity in two in vitro angiogenesis assays: the
endothelial cell proliferation and the chick aortic ring
assays.
Bovine Capillary Endothelial (BCE) Cell Proliferation
To determine if HPMA copolymer-TNP-470 was active in endothelial
cells we tested its inhibitory effect on BCE cell proliferation in
vitro. BCE cell growth, stimulated by bFGF, was inhibited similarly
by TNP-470 and HPMA copolymer-TNP-470 (FIG. 2A). Both free and
conjugated TNP-470 inhibited bFGF-induced proliferation.
(cytostatic effect) of BCE cells from 10 pg/ml to 1 .mu.g/ml
TNP-470-equivalent concentration. However, at doses higher than 1
.mu.g/ml both free and conjugated TNP-470 were cytotoxic. These
data are in agreement with published results of free TNP-470 on
different endothelial cells.sup.11,32.
Chick Aortic Ring Assay
Having demonstrated that the conjugate inhibited in vitro
endothelial cell growth, an ex-vivo model of chick aortic rings
implanted in Matrigel was utilized to further characterize the HPMA
copolymer-TNP-470 conjugate. Both free and conjugated TNP-470
reduced the number and length of vascular sprouts growing from the
chick aortic ring at 50 pg/ml and completely prevented outgrowth at
100 pg/ml (FIG. 2B). A control aortic ring (left panel) showed
abundant sprouting. Similar dose dependency was found for free
TNP-470 in a mouse aortic ring assay (Moulton, unpublished
results).
Hepatectomy
We have shown that HPMA copolymer-TNP-470 was equally-active as the
free TNP-470 in vitro. Therefore, we evaluated its antiangiogenic
activity in vivo.
Before testing the conjugate in tumor models in vivo, we
established the efficacy of HPMA copolymer-TNP-470 conjugate in the
hepatectomy model (FIG. 3A). This non-neoplastic model is a
relatively fast (8 days) in vivo angiogenesis-dependent
process.sup.33. We employed the hepatectomy model to compare the
endothelial cell inhibitory activity of free and conjugated
TNP-470, because liver regeneration post hepatectomy is
angiogenesis-dependent, similar to tumor growth.sup.33,34.
Following partial hepatectomy, control mice regenerated their
resected liver to their pre-operative mass (.about.1.2 g) by
post-operative day 8 (FIG. 3B). In mice treated subcutaneously
(s.c.) with free TNP-470 or its polymer-conjugated form at 30 mg/kg
every other day (q.o.d), the regeneration of the liver was
inhibited and livers reached the average size of 0.7 g on
post-operative day 8 (FIG. 3B). Free TNP-470 did not inhibit liver
regeneration when injected at 60 mg/kg every four days or at a
single injection of 120 mg/kg at the day of the hepatectomy.
However, HPMA copolymer-TNP-470 conjugate had an equivalent effect
as the 30 mg/kg q.o.d. dosing schedule when given every 4 days
(q.4.d.) at 60 mg/kg or at a single dose of 120 mg/kg on the day of
hepatectomy. This suggests that the conjugate has a longer
circulation time than the free TNP-470 in vivo and/or that the
conjugate accumulates at the site of proliferating endothelial
cells, leading to sustained release of TNP-470 from the polymer.
Because liver regeneration is regulated by endothelial
cells.sup.33,34, it was expected that a similar effect would occur
with proliferating endothelial cells in tumor tissue, where the
conjugate accumulates due to the EPR effect.
Early Mouse Development
Free and conjugated TNP-470 were injected into 7 and 17 day-old
BALB/c mice in order to test their effects on normal development.
Free TNP-470 inhibited growth, by inhibiting weight gain at this
critical age. However, HPMA copolymer-TNP-470 conjugate-treated
mice developed similarly to the control group injected with saline
(FIG. 3C). These results differed from the results obtained from
the hepatectomy experiments. HPMA copolymer-TNP-470 conjugate
inhibited liver regeneration following hepatectomy but did not
inhibit normal development in the newborn mice. A possible
explanation is that the conjugate extravasated through leaky
vessels in the liver following surgery (i.e., same inhibition as
seen in wound healing delayed by TNP-470 .sup.35). However, the
conjugate did not leak from normal vessels developing in the
newborn.
Evaluation of Antitumor Activity of HPMA Copolymer-TNP-470 on SCID
Mice Bearing s.c. A2058 Human Melanoma
Mice bearing s.c. A2058 melanoma showed increased survival when
treated with free and conjugated TNP-470 (T/C=0.34 for TNP-470 and
0.12 for the conjugate) (FIG. 4A). T/C was defined as the ratio of
the mean volume of tumor of the treated animals (T) divided by the
mean volume of tumor of the untreated control group (C). During
this study there were neither deaths due to toxicity nor weight
loss in the mice treated with the conjugate, indicating dose
escalation of the conjugate to be possible. A significant decrease
in tumor growth rate was observed in animals treated with TNP-470
(P<0.03) and with HPMA copolymer-TNP-470 (P<0.05) compared to
controls (FIG. 4A, B, C). FIG. 4C presents histological sections of
tumors representing the three treated groups (saline, free or
conjugated TNP-470) stained with H & E and showing viable tumor
cells in all.
Evaluation of Antitumor Activity of HPMA Copolymer-TNP-470 on
C57BL/6J Mice Bearing s.c. LLC
Mice bearing s.c. LLC showed increased survival when treated with
free and bound TNP-470 at equivalent concentration of TNP-470 of 30
mg/kg q.o.d. HPMA copolymer-TNP-470 exhibited superior antitumor
activity compared to free TNP-470. On day 8, when control mice were
sacrificed, HPMA copolymer-TNP-470 inhibited tumor growth by 86%
(P<0.03) whereas free TNP-470 by 67% (P<0.05) (FIG. 5A, B).
In addition, the conjugate did not induce weight loss whereas free
TNP-470 did (data not shown). Since HPMA copolymer-TNP-470 did not
induce weight loss, we tested the conjugate in LLC-bearing mice at
the higher doses of 60 and 90 as well as 30 mg/kg/q.o.d. The
conjugate inhibited tumor growth equally at 30 or 60 mg/kg/q.o.d
(P<0.03, T/C=0.4, day 8). Tumor suppression was significantly
enhanced at 90 mg/kg/q.o.d (P<0.05, T/C=0.24, day 8) (FIG. 5C,
D). Even at the higher dose of 90 mg/kg/q.o.d., there was no animal
weight loss, indicating we did not reach the maximum tolerated dose
(MTD). Free TNP-470 at these doses is known to be toxic to the
mice. In this set of experiments treatment was started when tumors
reached 500 mm.sup.3, therefore results differed from previous
experiments where treatment started when tumors were 100
mm.sup.3.
Evaluation of TNP-470 and HPMA Copolymer-TNP-470 in the
Cerebrospinal Fluid of Mice Bearing s.c. LLC
HPLC-Mass spectrometry (LC-MS/MS) showed that free TNP-470 is
present in the cerebrospinal fluid (CSF) of mice with s.c. LLC
tumor following i.v. administration of the drug. However, when HPMA
copolymer-TNP-470 conjugate was injected, neither TNP-470 nor its
known metabolites.sup.36 were detected in the CSF. These results
suggest that TNP-470-related neurotoxicity could be avoided when
TNP-470 is conjugated to HPMA copolymer. Full body distribution and
pharmacokinetics of free and conjugated TNP-470 in normal tissues,
blood, urine and tumor analyzed by LC-MS/MS will be published
separately.
Conclusions
Although a new departure in cancer therapy, several polymer-drug
conjugates are already in early clinical trials.sup.37. These
include HPMA copolymer-doxorubicin (PK1, FCE28068), 4PMA
copolymer-paclitaxel (PNU 166945), HPMA copolymer-camptothecin,
polyethylene glycol (PEG)-camptothecin, polyglutamic
acid-paclitaxel, an HPMA copolymer-platinate (AP5280) and also an
HPMA copolymer-doxorubicin conjugate bearing additionally
galactosamine (PK2, FCE28069).sup.38. Reduced toxicity and activity
in chemotherapy refractory patients has been described. In phase I,
PK1 displayed a maximum tolerated dose of 320 mg/m.sup.2 (compared
to 60 mg/m.sup.2 : for free doxorubicin) and also showed antitumor
activity.sup.39. Moreover, the clinical pharmacokinetics (PK1
t.sub.1/2a =1.8 h with no dose dependency of clearance compared to
few minutes for free doxorubicin) were very similar to those
reported in animals.sup.25. PK1 has proven ability to target solid
tumors by the EPR effect.sup.40 with concomitant renal elimination
resulting in low blood levels within 1-5 h in animals and in
humans.sup.25,39.
Polymer-angiogenesis inhibitor conjugates can capitalize on the
ability of macromolecules to target solid tumor tissue passively by
the EPR effect.sup.26 (similar to PK1). This effect occurs due to
the poorly organized tumor vasculature.sup.41 resulting in
`enhanced permeability` towards circulating molecules. The poor
lymphatic drainage in tumor tissue leads to increased `retention`.
It is accepted that the main reason for the improved antitumor
activity of the polymer-drug conjugates, with respect to the free
drug, is tumor targeting as a result of this EPR effect.sup.37.
Gly-Phe-Leu-Gly (SEQ ID NO: 1) polymer-TNP-470 linker used in this
study was designed to permit intralysosomal TNP-470 liberation due
to action of the lysosomal cysteine proteases.sup.29. In order to
exert an antitumor effect, an active TNP-470 species must be
released at the tumor site and interact with methionine
aminopeptidase 2 (MetAP2) in endothelial cells. MetAP2 is one
molecular target of TNP-470 that was recently identified, although
the precise mechanism underlying its selective effect on the
proliferation of endothelial cells is yet to be understood.sup.42.
Therefore, the T/C values for the conjugate of 0.12-0.14 indicated
that TNP-470, which was bound to the polymeric backbone during
circulation, was released at the tumor site. The mechanism for
release of a TNP-470 moiety involves cellular uptake, followed by
enzymatic cleavage of the peptide linker within the lysosomes of
endothelial cells. It is likely that some of the conjugate that
accumulates in the tumor will be taken up by tumor cells. However,
a higher concentration of TNP-470 will be needed to affect tumor
cells (3-logs higher).
Many studies of angiogenesis inducers and inhibitors rely on in
vitro or in vivo models as indicators of efficacy. However, as
valuable as these models are, there are limitations to each one of
these. Therefore, multiple assays used, involving both in vitro and
in vivo assays, are at present the best way to minimize the
problems inherent in any specific assay.sup.43. In this way, a
proper evaluation and comparison between free and conjugated
TNP-470, was achieved.
In summary, we have shown that tumor growth rate can be
significantly reduced by systemic delivery of an antiangiogenic
agent that is targeted to the tumor vasculature. In addition, this
conjugate likely leads to reduced toxicity and does not cause
weight loss in newborn and adult mice because, unlike the free
form, it does not enter the CSF. The enhanced and long acting
effect of the conjugate compared to that of the free TNP-470 (as
described in the hepatectomy model), can be ascribed to increased
accumulation in neovascularized tissues and to greater stability of
the conjugate. Several components of this strategy contribute to
its pronounced antitumor activity, which may facilitate future
therapy in humans. First, the HPMA copolymer used in this study has
multivalent side-chains, which make it possible to target high
loading of TNP-470 or other drugs to angiogenic blood vessels due
to the EPR effect. Second, it is feasible to conjugate an
endothelial cell targeting moiety to those side-chains on the
polymeric backbone.sup.44. Third, we emphasize that; (a)
angiogenesis inhibitors suppress endothelial growth from inside the
vascular lumen and may also traverse leaky tumor vessels; (b) the
conjugate HPMA copolymer-TNP-470 provides prolonged exposure of the
drug to endothelium; and (c) the conjugated TNP-470 cannot cross
normal blood brain barrier. Lastly, polymers are less immunogenic
than viral vectors and are known to decrease or even abrogate
immunogenicity of bound proteins and to prolong circulation
time.sup.24,45. Polymer-enzyme conjugates such as polyethylene
glycol (PEG)-L-asparaginase (Oncaspar.RTM.) for the treatment of
acute lymphoblastic leukemia have been FDA approved and has become
commercially avaliable.sup.46. Therefore, it may be feasible to
deliver therapeutic genes or proteins repeatedly to angiogenic
blood vessels for sustained treatment of diseases that depend on
angiogenesis and vascular remodeling. This study represents an
example of how an effective angiogenesis inhibitor can be
significantly improved and its toxicity decreased by conjugating it
to a polymer.
EXAMPLE 2
Miles Assay:
One of the problems with angiogenesis-dependent diseases is
increased vessel permeability (due to high levels of VPF) which
results in edema and loss of proteins. A decrease in vessel
permeability is beneficial in those diseases. We have found, using
the Miles assay (Claffey et al., Cancer Res., 56: 172-181 (1996)),
that free and bound TNP-470 block permeability. Briefly, a dye,
Evans Blue, was injected i.v. to anesthesized mice. After 10
minutes, human recombinant VEGF.sub.165 was injected intradermally
into the back skin. Leakage of protein-bound dye was detected as
blue spots on the underside of the back skin surrounding the
injection site. After 20 minutes, mice were euthanized. Then, the
skin was excised, left in formamide for 5 days to be extracted and
the solution read at 620 nm. Putative angiogenesis inhibitors such
as free and conjugated TNP-470 were injected daily 3 days prior to
the VEGF challenge. The same was repeated on tumor-bearing mice to
evaluate the effect of angiogenesis inhibitors on tumor vessel
permeability.
We have compared free and conjugated TNP-470 to other angiogenesis
inhibitors in the Miles assay. We have found that free TNP-470 and
HPMA copolymer-TNP-470 had similar inhibitory effect on VEGF
induced vessel permeability as opposed to the control groups and
indirect angiogenesis inhibitors such as Herceptin and Thalidomide
(FIG. 6).
EXAMPLE 3
TNP-470 and HPMA Copolymer-TNP 470 Selectively Inhibit Endothelial
Cell Proliferation
TNP-470 inhibited serum-induced proliferation (cytostatic effect)
of A2058 melanoma cells beginning at 10 ng/ml (FIG. 7). At doses
higher than 100 .mu.g/ml TNP-470 was cytotoxic to these cells.
TNP-470 was thus 4-logs more potent on endothelial cells than on
tumor cells. On both cell lines, HPMA copolymer-TNP-470 conjugate
had a similar effect on cell proliferation as the free TNP-470
(FIG. 7). HPMA copolymer alone was inert in vitro and in vivo (data
not shown), in agreement with extensive data previously published
on HPMA copolymers (reviewed in.sup.51).
Once Weekly Administration of HPMA Copolymer-TNP-470 Conjugate
Inhibits Angiogenisis in the Liver Regeneration Model
Free TNP-470 did not inhibit liver regeneration when injected at 60
mg/kg every four days or at a single injection of 120 mg/kg at the
day of hepatectomy. However, HPMA copolymer-TNP-470 conjugate had
an equivalent effect as the 30 mg/kg q.o.d. dosing schedule when
given every 4 days (q.4.d.) at 60 mg/kg or at a single dose of 120
mg/kg on the day of hepatectomy. This suggests that the conjugate
has a longer circulation time than the free TNP-470 in vivo and/or
that the conjugate accumulates at the site of proliferating
endothelial cells, leading to sustained release of TNP-470 from the
polymer.
HPMA Copolymer-TNP-470 Conjugate Accumulates at Higher
Concentration in Tumors and has a Longer Half-life in the
Circulation than Free TNP-470
Free TNP-470 concentrations from serum specimens were only detected
at the 1 and 2 h time points; with mean concentrations of 0.9, 1.7
ng/ml, respectively. There was no detectable TNP-470 in serum after
then 2 h time point. Furthermore, no detectable concentrations of
free TNP-470 were observed form tumor specimens at any given sample
time points. However, TNP-470 active species, extracted sera and
tumors of mice injected with HPMA copolymer-TNP-470, were present
up to 48 h post injection (FIG. 8a and FIG. 8b). Half-life of
circulating serum of the HPMA copolymer-TNP-470 mice sera is
estimated up to 24 hours.
HPMA Copolymer-TNP-470 does not Affect Neurological Function
It has been shown that TNP-470 treatment results in severe ataxia
and other symptoms of cerebellar dysfunction in humans.sup.52.
Therefore, we tested the effects of TNP-470 and HPMA
copolymer-TNP-470 on the motor skills of mice using the rotorod
test, a classic assay for ataxia in rodents. Mice are placed on a
rod that rotates at increasing speed, and the time that the mice
remain on the rod is recorded (FIG. 9a). The performance of animals
injected with HPMA copolymer-TNP-470 was indistinguishable from
that of control mice, while mice injected with free TNP-470
remained on the rotating rod for significantly shorter times than
the other 2 groups (P<0.03) (FIG. 9b). The experiment was
repeated every day for 5 consecutive days with similar results
(data not shown). Mice treated with free TNP-470 lost weight, while
mice treated with HPMA copolymer-TNP-470 gained weight similar to
control mice (P<0.01) (FIG. 9c). These results indicate that
while TNP-470 injection leads to ataxia, HPMA copolymer-TNP-470
does not affect the motor coordination of mice. Interestingly,
there were no visible neurohistological alterations in the mice
injected with free TNP-470 (data not shown). This indicates that
free TNP-470 induces neuronal dysfunction but does not affect
neuronal survival, consistent with the observation that the
neurological side effects in humans are reversible.sup.52.
Conclusion
Several polymer-cytotoxic drug conjugates are already in early
clinical trials.sup.53. These include HPMA copolymer-doxorubicin
(PK1, FCE28068), HPMA copolymer-paclitaxel (PNU 166945),HPMA
copolymer-camptothecin, polyethylene glycol (PEG)-camptothecin,
polyglutamic acid-paclitaxel, and HPMA copolymer-platinate (AP5280)
and also an HPMA copolymer-doxorubicin conjugate bearing
additionally galactosamine as a targeting moiety to the liver (PK2,
FCE28069).sup.51. Reduced toxicity and activity in chemotherapy
refractory patients has been described. In phase I, PK1 displayed a
maximum tolerated dose of 320 mg/m.sup.2 (compared to 60 mg/.sup.2
for free doxorubicin) and also showed antitumor activity.sup.54.
Moreover, the clinical pharmacokinetics (PK1 t.sub.1/2a =1.8 h with
no dose dependency of clearance compared to a few minutes for free
doxorubicin) were very similar to those reported in animals.sup.55.
PK1 has proven ability to target solid tumors by the EPR
effect.sup.56 with concomitant renal elimination resulting in low
blood levels within 1-5 h in animals and in humans.sup.54,55.
In order to exert an antitumor effect, an active TNP-470 species
must be released at the tumor site and interact with methionine
aminopeptidase 2 (MetAP2) in endothelial cells. MetAP2 is one
molecular target of TNP-470 that was recently identified, although
the precise mechanism underlying its selective effect on the
proliferation of endothelial cells is yet to be understood.sup.57.
Therefore, the T/C values for the conjugate of 0.12-0.14 indicated
that TNP-470, which was bound to the polymeric backbone during
circulation, was released at the tumor site in an active form. The
mechanism for release of a TNP-470 moiety involves cellular uptake,
followed by enzymatic cleavage of the peptide linker within the
lysosomes of endothelial cells. It is likely that some of the
conjugate that accumulates in the tumor will be taken up by tumor
cells.
There are two main reasons why the conjugate should affect
endothelial cells in tumors and regenerating livers, but not affect
those of the neonate and the blood brain barrier and other
quiescent vessels. The first reason is that TNP-470 only affects
proliferating endothelial cells. TNP-470 is known to induce p53
activation through a unique mechanism in endothelial cells leading
to an increase in cyclin-dependent kinase inhibitor p21.sup.CIP/WAF
expression and subsequent growth arrest.sup.58,59. p21 prevents the
entry of the cells in S phase by inhibiting the activity of CDK2.
Jing-Ruey et al., showed that TNP-470 selectively arrests the
growth of endothelial cells, but not non-endothelial cells by
activating p53 and inducing p21 only in endothelial cells. Further
more, Zhang et al. showed that TNP-470 did not affect
contact-inhibited endothelial cells in the G0-G1 phase.
The liver endothelial cells are sensitive to TNP-470 in our
experiment only during the 8 day period of endothelial
proliferation in regenerating liver.sup.60,61. In contrast, the
endothelial cells lining the blood brain barrier are not
proliferating. However, the unresponsiveness of quiescent,
non-proliferating endothelial cells lining the blood brain barrier
does not prevent the diffusion of free TNP-470 to the brain tissue.
The most likely mechanism by which free TNP-470 is neurotoxic is by
directly affecting neuronal function. However, the mechanism for
this is still unknown. The fact that the neurological effects of
free TNP-470 in humans are rapidly reversible upon discontinuation
of TNP-470 treatment suggests that TNP-470 does not produce
long-term neuronal degeneration.
The second reason that the conjugate shows selective effects
against tumor and regenerating liver endothelial cells, is that the
conjugate circulates for a longer time than TNP-470 and accumulates
selectively at higher concentration in tissues where vessels are
leaky. On the other hand, free TNP-470 can diffuse from normal
vessels homogenously throughout the body. Hence, the proliferating
endothelial cells in the leaky environments of the tumor and the
regenerating liver will be exposed for a much longer time to
TNP-470 when it is conjugated to the polymer due to its size and
structure (enhanced permeability and retention (EPR) effect).
Seymour et al., have shown that the HPMA copolymer conjugates are
internalized into cells via slow fluid phase pinocytosis.sup.62.
Hence, these HPMA conjugates need to be present in the vessel
microenvironment for a period of time in order to internalize into
the endothelial cells. In short, HPMA-TNP-470 conjugate requires
both the leaky environment and proliferating endothelial cells to
be effective.
The references cited throughout the specification are incorporated
herein by reference.
References 1. Folkman, J. Angiogenesis. in Harrison's Textbook of
Internal Medicine (eds. Braunwald, E. et al.) 517-530 (McGraw Hill,
New York, 2001). 2. Hanahan, D. & Folkman, J. Patterns and
emerging mechanisms of the angiogenic switch during tumorigenesis.
Cell 86, 353-64 (1996). 3. Volpert, O. V. et al. Id1 regulates
angiogenesis through transcriptional repression of
thrombospondin-1. Cancer Cell 2, 473-483 (2002). 4. Folkman, J.
Tumor angiogenesis. in Cancer Medicine (eds. Holland, J. et al.)
132-152 (B. C. Decker Inc., Ontario, Canada, 2000). 5. Lyden, D. et
al. Id1 and Id3 are required for neurogenesis, angiogenesis and
vascularization of tumour xenografts. Nature 401, 670-7 (1999). 6.
Streit, M. et al. Thrombospondin-2: a potent endogenous inhibitor
of tumor growth and angiogenesis. Proc Natl Acad Sci USA 96,
14888-93 (1999). 7. Chin, L. et al. Essential role for oncogenic
Ras in tumour maintenance. Nature 400, 468-72 (1999). 8. Tabone, M.
D. et al. Are basic fibroblast growth factor and vascular
endothelial growth factor prognostic indicators in pediatric
patients with malignant solid tumors? Clin Cancer Res 7, 538-43
(2001). 9. Yao, Y. et al. Prognostic value of vascular endothelial
growth factor and its receptors Flt-1 and Flk-1 in astrocytic
tumours. Acta Neurochir (Wien) 143, 159-66 (2001). 10. Yuan, A. et
al. Aberrant p53 expression correlates with expression of vascular
endothelial growth factor mRNA and interleukin-8 mRNA and
neoangiogenesis in non-small-cell lung cancer. J Clin Oncol 20,
900-910 (2002). 11. Ingber, D. et al. Synthetic analogues of
fumagillin that inhibit angiogenesis and suppress tumour growth.
Nature 348, 555-7 (1990). 12. Antoine, N. et al. AGM-1470, a potent
angiogenesis inhibitor, prevents the entry of normal but not
transformed endothelial cells into the G1 phase of the cell cycle.
Cancer Res 54, 2073-6 (1994). 13. Folkman, J. Tumor angiogenesis.
in Accomplishments in cancer research (eds. Wells, S. J. &
Sharp, P.) 32-44 (Lippincott Williams & Wilkins, New York,
1998). 14. Kudelka, A. P., Verschraegen, C. F. & Loyer, E.
Complete remission of metastatic cervical cancer with the
angiogenesis inhibitor TNP-470. N Engl J Med 338, 991-2 (1998). 15.
Kudelka, A. P. et al. A phase I study of TNP-470 administered to
patients with advanced squamous cell cancer of the cervix. Clin
Cancer Res 3, 1501-5 (1997). 16. Bhargava, P. et al. A Phase I and
pharmacokinetic study of TNP-470 administered weekly: to patients
with advanced cancer. Clin Cancer Res 5, 1989-95 (1999). 17.
Herbst, R. S. et al. Safety and pharmacokinetic effects of TNP-470,
an angiogenesis inhibitor, combined with paclitaxel in patients
with solid tumors: evidence for activity in non-small-cell lung
cancer. J Clin Oncol 20, 4440-7 (2002). 18. Kim, E. S. &
Herbst, R. S. Angiogenesis inhibitors in lung cancer. Curr Oncol
Rep 4, 325-33 (2002). 19. Stadler, W. M. et al. Multi-institutional
study of the angiogenesis inhibitor TNP-470 in metastatic renal
carcinoma. J Clin Oncol 17, 2541-5 (1999). 20. Logothetis, C. J. et
al. Phase I trial of the angiogenesis inhibitor TNP-470 for
progressive androgen-independent prostate cancer. Clin Cancer Res
7, 1198-203 (2001). 21. Rupnick, M. A. et al. Adipose tissue mass
can be regulated through the vasculature. Proc Natl Acad Sci USA
99, 10730-5 (2002). 22. Schoof, D. D. et al. The influence of
angiogenesis inhibitor AGM-1470 on immune system status and tumor
growth in vitro. Int J Cancer 55, 630-5 (1993). 23. Nagabuchi, E.,
VanderKolk, W. E., Une, Y. & Ziegler, M. M. TNP-470
antiangiogenic therapy for advanced murine neuroblastoma. J.
Pediatr Surg 32, 287-93 (1997). 24. Rihova, B. et al.
Biocompatibility of N-(2-hydroxypropyl) methacrylamide copolymers
containing adriamycin. Immunogenicity, and effect on haematopoietic
stem cells in bone marrow in vivo and mouse splenocytes and human
peripheral blood lymphocytes in vitro. Biomaterials 10, 335-42.
(1989). 25. Seymour, L. W., Ulbrich, K., Strohalm, J., Kopecek, J.
& Duncan, R. The pharmacokinetics of polymer-bound adriamycin.
Biochem Pharmacol 39, 1125-31 (1990). 26. Maeda, H., Wu, J., Sawa,
T., Matsumura, Y. & Hori, K. Tumor vascular permeability and
the EPR effect in macromolecular therapeutics: a review. J Control
Release 65, 271-84 (2000). 27. Duncan, R., Coatsworth, J. K. &
Burtles, S. Preclinical toxicology of a novel polymeric antitumour
agent: HPMA copolymer-doxorubicin (PK1). Hum Exp Toxicol 17, 93-104
(1998). 28. Satchi-Fainaro, R. Targeting tumour vasculature:
Reality or a dream? J Drug Targeting 10, 529-533 (2002). 29.
Duncan, R., Cable, H. C., Lloyd, J. B., Rejmanova, P. &
Kopecek, J. Polymers containing enzymatically degradable bonds, 7.
Design of oligopeptide side chain in poly
N-(2-hydroxypropyl)methacrylamide copolymers to promote efficient
degradation by lysosomal enzymes. Makromol Chem 184, 1997-2008
(1984). 30. Foekens, J. A. et al., Prognostic significance of
cathepsins B and L in primary human breast cancer. J Clin Oncol 16,
1013-21 (1998). 31. Gianasi, E. et al. HPMA copolymer platinates as
novel antitumour agents: in vitro properties, pharmacokinetics and
antitumour activity in vivo. Eur J Cancer 35, 994-1002 (1999). 32.
Kusaka, M. et al. Cytostatic inhibition of endothelial cell growth
by the angiogenesis inhibitor TNP-470 (AGM-1470). Br J Cancer 69,
212-6 (1994). 33. Greene, A. K. et al. Endothelial directed hepatic
regeneration after partial hepatectomy. Annals of Surgery in press
(2003). 34. Drixler, T. A. et al. Liver regeneration is an
angiogenesis-associated phenomenon. Ann Surg 236, 703-12 (2002).
35. Klein, S. A., Bond, S. J., Gupta, S. C., Yacoub, O. A. &
Anderson, G. L. Angiogenesis inhibitor TNP-470 inhibits murine
cutaneous wound healing. J Surg Res 82, 268-74 (1999). 36. Whalen,
C. T., Hanson, G. D., Putzer, K. J., Mayer, M. D. & Mulford, D.
J. Assay of TNP-470 and its two major metabolites in human plasma
by high-performance liquid chromatography-mass spectrometry. J
Chromatogr Sci 40, 214-8 (2002). 37. Brocchini, S. & Duncan, R.
Polymer-Drug conjugates: drug release from pendent linkers. in
Encyclopaedia of controlled release (ed. Mathiovitz, E.) 786-816
(New York: Wiley, 1999). 38. Duncan, R. et al. Polymer-drug
conjugates, PDEPT and PELT: basic principles for design and
transfer from the laboratory to clinic. J Control Release 74,
135-46 (2001). 39. Vasey, P. A. et al. Phase I clinical and
pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide
copolymer doxorubicin]: first member of a new class of
chemotherapeutic agents-drug-polymer conjugates. Cancer Research
Campaign Phase I/II Committee. Clin Cancer Res 5, 83-94 (11999).
40. Seymour, L. W. et al. Tumour tropism and anti-cancer efficacy
of polymer-based doxorubicin prodrugs in the treatment of
subcutaneous murine B16F10 melanoma. Br J Cancer 70, 636-41 (1994).
41. Dvorak, H. F., Nagy, J. A., Dvorak, J. T. & Dvorak, A. M.
Identification and characterization of the blood vessels of solid
tumors that are leaky to circulating macromolecules. Am J Pathol
133, 95-109 (1988). 42. Griffith, E. C. et al. Methionine
aminopeptidase (type 2) is the common target for angiogenesis
inhibitors AGM-1470 and ovalicin. Chem Biol 4, 461-71 (1997). 43.
Auerbach, R., Akhtar, N., Lewis, R. L. & Shinners, B. L.
Angiogenesis assays: problems and pitfalls. Cancer Metastasis Rev
19, 167-72 (2000). 44. Seymour, L. W. et al. Hepatic drug
targeting: phase I evaluation of polymer-bound doxorubicin. J Clin
Oncol 20, 1668-76 (2002). 45. Francis, G. E., Delgado, C. &
Fisher, D. PEG-modified proteins. in Stability of proteins
pharmaceuticals (Part B) (ed. Ahem T J, M. M.) 235-263 (Plenum
Press, New York, 1992). 46. Ho, D. H. et al. Clinical pharmacology
of polyethylene glycol-L-asparaginase. Drug Metab Dispos 14, 349-52
(1986). 47. O'Reilly, M. S. et al. Angiostatin: a novel
angiogenesis inhibitor that mediates the suppression of metastases
by a Lewis lung carcinoma. Cell 79, 315-28 (1994). 48. Folkman, J.,
Haudenschild, C. C. & Zetter, B. R. Long-term culture of
capillary endothelial cells. Proc Natl Acad Sci USA 76, 5217-21
(1979). 49. Paxinos, J. & Franklin, K. B. J. The Mouse Brain in
Stereotaxic Coordinates, (Academic Press, 2001). 50. Waynforth, H.
B. Routes and methods of administration, Intracerebral injection.
in Experimental and Surgical technique in the rat, Vol. 2.9 34-36
(Academic Press, London, 1980). 51. Duncan, R et al. Polymer-drug
conjugates, PDEPT and PELT: basic principles for design and
transfer from the laboratory to clinic. J Control Release 74,
135-46 (2001). 52. Bhargava, P. et al. A Phase I and
pharmacokinetic study of TNP-470 administered weekly to patients
with advanced cancer. Clin Cancer Res 5, 1989-95 (1999). 53.
Brocchini, S. & Duncan, R. Polymer-Drug conjugates: drug
release from pendent linkers. In Encyclopaedia of controlled
release (ed. Mathiovitz, E.) 786-816 (New York: Wile, 1999). 54.
Vasey, P. A. et al. Phase 1 clinical and pharmacokinetic study of
PK1 [N-2(2-hydroxypropyl) methacrylamide copolymer doxorubicin]:
first member of a new class of chemotherapeutic agents-drug-polymer
conjugates. Cancer Research Campaign Phase I/II Committee. Clin
Cancer Res 5, 83-94 (1999). 55. Seymour, L. W., Ulbrich, K.,.
Strohalm, J., Kopecek, J. & Duncan, R. The pharmacokinetics of
polymer-bound adriamycin. Biochem Pharmacol 39, 1125-31 (1990). 56.
Seymour, L. W. et al. Tumour tropism and anti-cancer efficacy of
polymer-based doxorubicin prodrugs in the treatment of subcutaneous
murine B16F10 melanoma. Br J Cancer 70, 636-41 (1994). 57.
Griffith, E. C. et al. Methionine aminopeptidase (type 2) is the
common target for angiogenesis inhibitors AGM-1470 and ovalicin.
Chem Biol 4, 461-71 (1997). 58. Yeh, J. R., Mohan, R. & Crews,
C. M. The antiangiogenic agent TNP-470 requires p53 and p21CIP/WAF
for endothelial cell growth arrest. Proc Natl Acad Sci USA 97,
12782-7 (2000). 59. Zhang, Y., Griffith, E. C., Sage, J., Jacks, T.
& Liu, J. O. Cell cycle inhibition by the anti-angiogenic
agent. TNP-470 is mediated by p53 and p21WAF1/CIP1. Proc Natl Acad
Sci USA 97 6427-32 (2000). 60. Greene, A. K. et al.
Endothelial-directed hepatic regeneration after partial
hepatectomy. Ann Surg 237, 530-5 (2003). 61. Drixler, T. A. et al.
Liver regeneration is an angiogenesis-associated phenomenon. Ann
Surg 236, 703-12 (2002). 62. Seymour, L. W. et al.
N-2(2-hydroxypropyl) methacrylamide copolymers targeted to the
hepatocyte galactose-receptor: pharmacokinetics in DBA2 mice. Br J
Cancer 63, 859-66(1991).
Although the foregoing invention has been described in some detail
by way of illustration and example for the purposes of clarity of
understanding, one skilled in the art will easily ascertain that
certain changes and modifications may be practiced without
departing from the spirit and scope of the appended claims.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 1 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence
Synthetic linker sequence <400> SEQUENCE: 1 Gly Phe Leu Gly
1
* * * * *